汉滩病毒囊膜糖蛋白G_2基因重组腺病毒的构建与表达

获得汉滩病毒G2 基因 ,构建其重组腺病毒并在HEK2 93细胞中包装表达 ,为研究汉滩病毒基因疫苗提供了实验基础。设计引物采用PCR从含汉滩病毒 \|76 1 1 8株M基因的M5 6质粒扩增出糖蛋白G2 基因片段 ,并将其克隆入腺病毒载体Adeno XviralDNA ,筛选获得重组腺病毒DNA ,转染HEK2 93细胞 ,包装、扩增后得到汉滩病毒G2 基因重组腺病毒原种 ;并在感染细胞内初步表达 ,用ELISA检测表达产物。得到了含汉滩病毒G2 基因的重组腺病毒 ,其滴度约为 1 0 10 pfu/mL ,同时在感染的HEK2 93细胞中检测到汉滩病毒糖蛋白G2 的表达。含汉滩病毒糖蛋白G2 基因重组腺病毒的成功构建 。

疏风解毒胶囊对奥密克戎无症状感染者及轻型患者舌象特征的影响

目的 探讨疏风解毒胶囊在隔离观察下对奥密克戎无症状感染者及轻型患者舌象变化特征的影响。方法采取前瞻性研究设计随机对照试验研究设计的方法,将2022年5月1日—2022年5月31日上海崇明岛复兴馆方舱医院收集的100例奥密克戎无症状感染者及轻型患者分为治疗组和对照组各50例。治疗组给予疏风解毒胶囊服用,同时给予患者隔离观察和常规护理,对照组患者给予隔离观察和常规护理。两组均观察治疗前及治疗后第3天(第1疗程)、第6天(第2疗程)的舌象变化与病情的变化,并对舌象与疗效进行分析。结果 治疗后第3天、第6天时,试验组在淡白舌的恢复效果上较对照组明显;试验组苔色(第3天、第6天)、苔质(第3天、第6天)及六经(第6天)较治疗前分布差异有统计学意义(P<0.05);对照组苔色(第6天)、苔质(第6天)及六经(第6天)分布差异有统计学意义(P<0.05),两组虽均有改善,但试验组改善情况显著优于对照组;对照组苔色(第3天)、苔质(第3天)及六经(第6天)分布差异无统计学意义(P>0.05),较治疗前无显著性改善。住院期间所有患者均未发生严重不良反应。结论疏风解毒胶囊治疗奥密克戎无症状感染者及轻型患者在舌象及六经分布的改善情况优于对照组,尤以治疗后第6天时改善更加明显,且安全性良好。

疏风解毒胶囊治疗奥密克戎无症状感染者及轻型患者临床分析

目的探讨疏风解毒胶囊在隔离观察下治疗奥密克戎无症状感染者及轻型患者临床疗效及舌象变化。方法将2022年4月—2022年6月上海崇明岛复兴馆方舱医院收集的100例无症状感染者及轻型奥密克戎患者采用随机分组法分为治疗组和对照组各50例。治疗组在隔离观察下给予疏风解毒胶囊加常规护理;对照组在隔离观察下给予常规护理。两组均观察治疗前及治疗3 d(第1疗程)、6 d(第2疗程)的临床症状改善情况。结果用疏风解毒胶囊常规治疗3 d后,发热、咳嗽及咽痛患者显著减少(P<0.05),且治疗组明显优于对照组;6 d后,发热、乏力、头痛、鼻塞、咽痛、咳痰及流涕的患者均较治疗前显著减少(P<0.05),且治疗组明显优于对照组。对照组核酸转阴时间中位数为9.0(8.0,11.0)d,平均住院时间中位数为12.0(11.0,14.0)d。治疗组核酸转阴时间中位数为7.0(6.0,8.0)d,平均住院时间中位数为10.0(9.0,11.0)d。住院期间所有患者均未发生严重不良反应事件。结论疏风解毒胶囊可有效改善奥密克戎无症状感染者及轻型患者临床症状,缩短核酸转阴时间及住院时间,且安全性好。

两种小菜蛾寄生蜂毒液器官超微结构的比较

应用超薄切片和电镜技术 ,观察比较了两种小菜蛾的内寄生蜂———小菜蛾盘绒茧蜂和半闭弯尾姬蜂毒液器官的形态结构。两种蜂的毒液器官均由一个毒囊和两条毒腺组成 ,但毒腺与毒囊的连接位点不同 ,毒腺分别接于毒囊的基部和顶端。它们的毒腺均由分泌细胞、退化的外胚层细胞和环腔的内膜三层结构构成 ,分泌细胞内含司分泌毒液的囊状细胞器及大量的内质网、线粒体、高尔基体、溶酶体和液泡。半闭弯尾姬蜂的分泌细胞内有分支的细胞核。小菜蛾盘绒茧蜂的毒囊由肌肉鞘和扁平细胞层构成 ,内膜呈波浪状内突并沿囊腔有几丁质增厚 ;半闭弯尾姬蜂的毒囊由肌肉鞘、分泌细胞和扁平细胞三层构成 ,分泌细胞基部内陷成一系列大的突起 ,这些结构与雌蜂的分泌和排出毒液的行为密切相关。在衰老的小菜蛾盘绒茧蜂的腺细胞中还存在大量的髓样体。本文就两种寄生蜂毒液器官的生物学、细胞生理学及相关的分类进化的意义进行了讨论。

管氏肿腿蜂雌性生殖系统解剖结构的研究

经解剖观察管氏肿腿蜂雌性生殖系统,得知其内部生殖系统由卵巢、侧输卵管、中输卵管、受精囊、毒囊及毒腺等组成;卵巢由左右两部分卵巢管组成,卵巢管总数为6～8条,单侧的卵巢管数为3～4条,两侧存在不对称现象;毒囊多呈葫芦状,中部缢缩处附着1对上端分支毒腺,分支数为1～7条不等。外生殖器由载瓣片、产卵瓣、产卵瓣鞘和已经特化为毒针的1对内产卵瓣等组成,相互呈嵌套结构。研究雌性生殖系统的形态结构,尤其是毒囊、毒腺等,对了解其寄生机理具有重要价值,且可为确定人工扩繁的商品蜂有效保藏期提供参考依据。

棒络新妇Nephila clavata粗毒的制备及毒理的初步研究

目的从整体生理效应上分析推测棒络新妇粗毒对无脊椎和脊椎动物运动系统的作用情况和影响机理。方法该实验研究内容及步骤包括:棒络新妇野外捕获、毒囊剥离法制备粗毒、昆虫腹部注射毒理实验、青蛙腿部肌肉注射毒理实验和青蛙坐股神经-腓肠肌电生理实验。结果实验表明棒络新妇粗毒是一种广谱动物运动阻断性毒素,既对无脊椎动物有影响,也能使脊椎动物产生反应;它不是通过影响神经传导组织发生作用的,而是直接作用于肌细胞。结论该实验采用毒囊剥离法制备的棒络新妇粗毒更加稳定有效,其粗毒作用机理有别于毒量较大的地穴型蜘蛛和毒性较强的结网型蜘蛛。

蜜蜂毒的生产技术

蜜蜂毒是工蜂毒囊分泌的一种毒汁,平时储存在毒囊中,当对敌人进行蜇刺时,毒汁通过蜇针的针管注射入敌体内,作为蜜蜂保卫家园的武器。蜂毒的成分十分复杂,含有众多的生物活性物质,是治疗风湿性关节炎等多种疾病的特效药物。新鲜的蜜蜂毒为浅黄色透明液体,气味清香,味苦,常温下容易挥发,易溶于水,在密封干燥的情况下,可以保存数年。由于蜜蜂毒在疾病治疗中疗效显著,而且数量较少,难于生产,蜜蜂毒的价格昂贵,开发研究和组织蜜蜂毒生产,可以大幅度地提高养蜂生产的经济效益。现将生产蜜蜂毒的方法介绍如下:一、乙醚麻醉法将蜜蜂集中抖入塑料袋中,根据蜜蜂数量的多少,放入沾乙醚的棉球,袋内还放一些干燥的吸水纸。当蜜蜂受到乙醚的刺激之后,就会将毒汁排到袋内的吸水纸上,然后取出吸水纸进行漂洗,经水分蒸发后,就可得到晶块状的蜂毒。袋内蜜蜂受到刺激排毒后,约2分钟,立即将蜜蜂取出,放在空气流通处,蜜蜂便慢慢苏醒而恢复活动。

凹纹胡蜂蜂毒中一种新型多肽的识别与活性检测

【目的】从凹纹胡峰蜂毒中识别出具有生物活性的新多肽,为后续相关药物的开发奠定基础。【方法】提取胡蜂毒囊中的RNA,构建cDNA文库,利用特殊引物筛选出目标基因,以此识别新多肽,并对新多肽的生物活性进行检测。【结果】得到新的凹纹胡蜂蜂毒多肽,并命名为VV-VP-2;其质量浓度为100~200μg/mL时对金黄色葡萄球菌(Staphylococcus aureus)有抑制作用,质量浓度为50~200μg/mL时对肺炎克雷伯菌(Klebsiella pneumoniae)有抑制作用。此外,5~200μg/mL的VV-VP-2对DPPH和ABTS自由基均有极显著的清除能力(P<0.01或P<0.001),50~200μg/mL的VV-VP-2对·OH自由基具有极显著的清除能力(P<0.01或P<0.001)。【结论】新多肽VV-VP-2具有开发成为抗菌和抗氧化药物的潜力,可为综合性药物或保健品的开发奠定基础。

刍议疏风解毒胶囊防治新型冠状病毒肺炎

新型冠状病毒肺炎(COVID-19),以发热、乏力、干咳为主要临床表现,具有强烈的传染性和流行性。结合中医传统理论,专家认为新冠肺炎属于"疫病"范畴,其病机为疫疠之邪犯肺,正邪相争,临床证候以寒湿为主,兼夹热毒,久则虚实夹杂。国家卫生健康委员会和国家中医药管理局联合推荐中西医结合防治新冠肺炎。疏风解毒胶囊(颗粒)具有清瘟解毒,宣肺泄热的功效,被推荐用于新冠肺炎医学观察期乏力伴发热或临床治疗初期。现代药理学研究显示疏风解毒胶囊具有抗病毒、抗炎和免疫调节等作用。该文主要从中医"疫病"理论、现代药理研究和临床研究等方面刍议疏风解毒胶囊防治新冠肺炎的理论依据。

Research on the Relationship between Three Isomers of Microcystins and Environmental Factors

[Objective] The relationship between three isomers of microcystins and environmental factors were studied in the fields.[Method] Three isomers of microcystins （MC-LR,RR and YR） from water of five sampling spots in a northern reservoir were observed for one year with High Performance Liquid Chromatography analytical method in order to study the relationship between three isomers and environmental factors.[Result] The three isomers of microcystins showed positive correlation with chlorophyll a;LR and YR isomers all had significant linear positive correlations with the water temperature,but the RR isomer showed no significant correlation with the water temperature;LR and YR isomers had relatively significantly correlativities with the contents of total nitrogen,nitrate nitrogen and organic nitrogen,while the RR isomer only showed a significant negative correlation with the content of nitrate nitrogen;LR and RR isomers both showed significant positive correlations with the contents of total phosphorus and organic phosphorus,while the phosphorus hardly affected the YR isomer and showed no evident correlation.[Conclusion] The relationship between three isomers of microcystins and environmental factors such as chlorophyll a,water temperature,nitrogen,phosphorus were studied and investigated the reasons,which might offered a reference for controlling the growth of blue algae in water and toxin synthesis.

Photochemical oxidation mechanism of microcystin-RR by p-n heterojunction Ag/Ag_2O-BiVO_4

Microcystin-RR（MC-RR）,a form of microcystin with two arginine moieties,is a cyanobacterial toxin that has been detected across a wide geographic range.It is a great concern globally because of its potential liver toxicity.Herein,the abilities of BiVO4,Ag-BiVO4,Ag2O-BiVO4 and Ag/Ag2O-BiVO4 to photocatalytically degrade MC-RR under visible-light irradiation（λ≥420 nm） were investigated and compared.The possible degradation pathways were explored through analysis of the reaction intermediates by high-performance liquid chromatography-mass spectrometry.The results showed that the presence of Ag^0 enhanced the photocatalytic efficiency of Ag/Ag2O-BiVO4 via a synergetic effect between Ag2O and Ag^0 at the p-n heterojunction.Moreover,the presence of Ag^0 also greatly promoted the adsorption of MC-RR on the photocatalyst surface.Toxicological experiments on mice showed that the toxicity of MC-RR was significantly reduced after photocatalytic degradation.

Genetic Analysis of the VP2 Hypervariable Region of Thirty-six Infectious Bursal Disease Virus Isolates in China during 2009-2012

[Objective] Infectious bursal disease （IBD） is a highly contagious immuno- suppressive disease caused by infectious bursal disease virus （IBDV）. IBDV is ge- netically prone to mutation, which results in challenges to the disease prevention and control. Thus, it is necessary to continuously monitor the prevalence of IBDV. [Method] 36 IBDVs were identified from ten provinces in China from 2009 to 2012. Partial fragments of VP2, including the hypervariable region （HVR）, from new iso- lates were sequenced and analyzed through comparisons with published sequences of IBDV, including 18 strains isolated previously by our lab and 24 reference strains from China and around the world. [Result] Phylogenetic analysis showed a co-exis- tence of IBDV strains belonging to classic, variant, attenuated, and very virulent IB- DV （wlBDV） in China. wlBDVs remain the predominant strains in China and the new subgroup was emerging. Alignment analysis revealed several distinct amino acid mutations that might be involved in virulence or antigenicity variation. [Conclu- sion] The results offered evolutionary clues showing the emerging trend of obvious variations and diversity of IBDV in major poultry-producing regions of China particu- larly in recent years. These findings will contribute to a better understanding of the genetic evolution of IBDV.

Genomic Sequencing and Molecular Characteristics of A Very Virulent Strain of Infectious Bursal Disease Virus Isolated in China

[Objective] The paper was to determine the genomic sequence of a very virulent strain of infectious bursal disease virus（IBDV）,and study its molecular characteristics.[Method] A very virulent strain（vvIBDV）（HLJ-0504） of infectious bursal disease virus（IBDV） with special characters was isolated in China and its genome was sequenced.[Result] Sequence analysis showed that segment A of HLJ-0504 was derived from vvIBDV,while segment B was from a distinct ancestor.The morbidity and mortality of HLJ-0504 was 100% and 86.7%to SPF chickens,respectively.[Conclusion] vvIBDV with distinct segment B were still circulating and the evolution of IBDV was diversified in China.Besides,it is hard to imagine that the virulence of IBDV is determined solely by segment A or B.

Study on the Knockout and the Soluble Prokaryotic Expression of VP5 Protein Transmembrane Region of IBDV

[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.

Effects of microcystin-LR on hippocampal N-acetylaspartate and neurobehaviors in rats

The effects of low-doses of microcystin-leucinearginine （ MC-LR ） exposure on neurobehaviors and N-acetylaspartate （NAA） expression in the hippocampus of rats were investigated. After male Sprague-Dawley （SD） rats were treated intra-gastrically with different doses of MC-LR for 90 d, the locomotor activity, spatial learning and memory function were evaluated in the rats after treatment using open field tests and Morris water maze tests. The results show that MC-LR exposure can lead to impairment of the spatial learning capacity and locomotor activity in rats at the dose of 2. 00 p,g/kg. The levels of NAA in the hippocampus were measured by magnetic resonance spectroscopy （MRI）. A significant decrease of NAA/Cr ratio （ P 〈 0. 05） was observed in the hippocampous. This study indicates that intra-gastrical exposure to low-doses of MC-LR has adverse effects on neuronal behavior and NAA levels in the hippocampous.

解毒胶囊诱导放疗耐受性乳腺肿瘤的再致敏机制研究

目的体内体外评估解毒胶囊含药血清对放疗耐受性乳腺肿瘤细胞的杀伤效果,探讨其潜在作用机制。方法将解毒胶囊制备成溶液给予大鼠连读灌胃1周制备含药血清,在细胞水平,利用MTT法测试该含药血清对正常及放疗耐受性乳腺肿瘤细胞的杀伤效果,获得其半数抑制剂量(IC50)。利用Western Blot方法检测DNA损伤通路因子MDM2、p53及Chk1的磷酸化水平。最后,利用裸鼠成瘤实验,观察不同浓度含药血清对放疗耐受肿瘤细胞成瘤的抑制效果。结果10%含药血清能够明显抑制正常细胞生长(P<0.01),但对放疗耐受乳腺肿瘤细胞无明显效果(P=0.06)。当浓度提升到20%左右时,能够显著影响此类细胞增殖(P<0.05)。与正常细胞相比,放疗耐受细胞的DNA损伤修复通路显著活化,MDM2、p53及Chk1的磷酸化水平均明显升高。含药血清处理后能够反转该通路的活化。相应地,给予放疗耐受肿瘤细胞诱导的体内肿瘤不同浓度的含药血清处理,能够剂量依赖的抑制肿瘤生长。结论含药血清可逆转放疗耐受细胞的DNA损伤修复能力,在一定水平可杀伤肿瘤。提示该血清可与临床乳腺肿瘤放疗相结合达到更好治疗乳腺癌的目的。

Acute Toxicity Test of Chinese Herbal Compound Tongmai Tangyanming Capsule

[Objective] This study was conducted to determine the safe dose of Tongmai Tangyanming capsule in clinic by evaluating its acute toxicity, so as to ensure clinical medication safety. [Method] The test was designed according to ＂Technique Requirements of Research of New Chinese Medicine＂ and ＂Guide for Research of New Chinese Medicine＂, mice were intragastrically administrated with Tongmai Tangyanming capsule at a maximum concentration and a maximum volume, and the acute toxic response of mice was observed, so as to determine medi- na lethal dose （LDso） and maximum administration dosage of Tongmai Tangyanming capsule. [Result] The LD^o could not be detected in the test, and the maximum administration dosage of mouse was calculated to be 112.104 g of dried herbs/（kg.d）, equivalent to 260 times of clinical dose of adult. [Conclusion] Chinese herbal compound Tongmai Tangyanming capsule has low acute toxicity, and its clinical dose is safe and reliable.

Immune Effect of Combination of Universal Molecular Adjuvant and IBDV Subunit

In order to evaluate the immune effect of the protein expressed by the universal vector pET-mLTA-CTLA-4 plus IBDV subunit, the fusion protein mLTA-CTLA-4 was expressed and purified. Protein toxicity tests were carried out on rabbits.The VP2 gene of infectious bursal virus was amplified by RT-PCR, and lately used for pET-VP2 construction. Ten-day-old free healthy chickens were chosen for a grouped test, including the mLTA-CTLA-4（at different doses） plus VP2 groups, IBDV living vaccine group and control group. Serum and mucosal samples were collected regularly and the neutralization titers of IgG and IgA were assayed, while an animal protection test was conducted to determine the protection rate. The results showed that the protein m LTA-CTLA-4 was non-toxic and its protection rate was100%. IgG or IgA levels in the IBDV vaccine group were slightly higher than those in recombinant protein groups. These results indicated that the recombinant protein mLTA-CTLA-4 could be applied with IBDV subunit vaccine to protect chickens from infection.

Comparison on the Tolerance of Cruciferous Crops and Leguminous Crops to Microcystin

[Objective] To study the differences in the tolerance of leguminous crops and cruciferous crops to microcystin （MC）. [Methed] The cruciferous typical crops oilseed rapes, pakchois, cabbages and leguminous typical crops soybeans, peas and broad beans were selected as the materials to test the effects of MC of differ- ent concentrations on the germination, growth and development of leguminous crops and cruciferous crops. The measurement indicators included germination rate, plant height, chlorophyll, etc. [Result] The MC had great effects on the oilseed rape and pakchois of cruciferous crops, and smaller effects on cabbage, while the leguminous crops were generally not affected. [Conclusion] Leguminous crops are more tolerant to MC than cruciferous crops and more preferential in MC polluted regions.